Project Summary Prior research demonstrates that afferent responses from semicircular canal cristae and otolith organ maculae deviate from the coherent mechanical stimulation imparted by their overlying accessory structures. This implicates further processing by hair cells (HCs), primary afferents, and the HC - afferent synapse. Processing is complicated by the parallel modes of synaptic transmission between HCs and afferents, and the convergence of multiple HCs onto a single afferent. In the vestibular periphery progress towards describing variations in afferent discharge in terms of the time course of underlying voltage- and ion-sensitive conductances has been impeded by two major anatomical features of the vestibular epithelia: 1) access to HCs and afferents in their native bi-ionic (endolymph - perilymph) environment is mechanically impeded, so there are few in situ recordings to serve as controls for pathophysiology in recordings made from isolated cells or epithelial explants; and 2) analysis of integration at the level of a ramifying afferent is complicated by multiple HC convergence onto each, and the impossibility of using a single patch-electrode to space-clamp a distributed afferent arbor. To overcome the inaccessibility problem associated with obtaining physiological data for vestibular HCs in their native environment, we will measure voltages using slow, potentiometric (Nernstian) dyes, whose equilibrium partition (concentration) is voltage-dependent. These dyes will be superfused across the vestibular and auditory epithelia in a turtle half-head preparation. The voltage-dependent fluorescence of slow redistributive dyes will be measured using multiphoton microscopy (MPM), and calibrated using microelectrode recordings from HCs, afferents and supporting cells via the readily accessible perilymphatic space of the auditory papilla. The type I HC/calyceal afferent synapse is relatively compact electrically, but HC convergence onto a single afferent over distances of 10s to 100s of microns makes it difficult to address the passive and active properties of an afferent arbor. As a consequence, it remains problematic to characterize afferent integration using conventional electrophysiological techniques. We will examine afferent convergence by patch recording single afferents using electrodes filled with electrochromic voltage-sensitive dyes (VSDs). Steady-state depolarizations and hyperpolarizations of the afferent via the patch electrode will be used to optically characterize the passive cable properties of the ramifying afferent using lattice light sheet microscopy (LLSM). Pulses of current injected through the patch electrode, or electrical stimulation of the nerve - phase-locked to image acquisition on the LLSM - will be used to image and average orthodromic and antidromic AP propagation in the parent axon and throughout the afferent arbor. We propose novel approaches to make highly significant measures that are currently unavailable. By using optical and electrode recordings to characterize the cellular potentials and the afferent convergence, these experiments have the potential to make large and durable contributions to the field.